Process for the production on an antiviral vaccine, particularly anti-foot and mouth disease vaccine

ABSTRACT

The instant invention relates to a process in which important improvements are introduced in virus production and inactivation steps, as well as in the vaccine formulation step. The virus is reproduced in animals immuno-depressed through ionizing radiations, thus making it possible to use adult host animals, not naturally sensitive to the virus, with the advantage of using all types of tissues thereof. Bacterial infections are prevented by antibiotic administration. Virus inactivation is also obtained through ionizing radiations. Radiation doses do not affect antigenic properties, as may be noted from the interpretation of the Target Theory.

BACKGROUND OF THE INVENTION--PRIOR ART

The instant invention relates to a novel process for preparing antiviralvaccines, particularly anti-foot and mouth disease vaccine.

As already known, the different processes for preparing vaccines againstviral diseases are comprised by a sequence of steps which, althoughdifferent in accordance with particular viruses and processes selected,may be classified as follows:

1. one or more steps for virus production;

2. one or more steps for virus inactivation; and

3. one or more steps for vaccine formulation.

The object of the present process invention has novel basic features invirus production and inactivation steps, and also proposes other novelsteps and complementary stages through the whole process as preferredalternatives.

In accordance with the present invention, virus production is higherthan that obtained through other known processes basically due to thefact that animals used are previously radiated with an ionizingradiation dose determined experimentally.

On the other hand, virus inactivation also is improved as compared tothat obtained through techniques of known processes, due to the factthat ionizing radiations are also used, which were experimentallydetermined. The reason why better results are obtained is that radiationis carried out over a viral suspension at a very low temperature (-40°C. or lower), thus avoiding virus degradation through the action ofenzymes present in the viral culture medium.

Foot and mouth disease virus easily reproduces in mice, rats, hamstersand rabbits not more than 8 days old. Sensitivity to virus decreases asthe age of the animals increases; thus, in adult animals, even highviral doses do not produce noticeable alterations.

However, it is important to use organs of adult animals for viralproduction in order to diminish production costs.

In accordance with the process of the instant invention virus productionis carried out in an animal previously subjected to immunodepressionthrough ionizing radiations, thus becoming sensitive to foot and mouthdisease virus even when adult, and in tissues which are normally notsensitive to the virus.

In prior art anti-foot and mouth disease vaccines virus production wascarried out by infecting bovine cattle, which are particularly sensitiveto the virus, and then removing the viral material present in the tongueepithelium.

The material thus obtained, besides being scarce, was subjected tofrequent contamination; therefore, vaccine mass production waspractically unfeasible due to the cost and operating difficulties.

Further, survival culture of bovine tongue epithelium obtained fromanimals killed in slaughterhouses as support for viral reproduction wasintroduced (Frankel method). This method is still now one of the mostwidely used in the industry for the reproduction of foot and mouthdisease virus for preparing vaccines.

Nevertheless, bacterial contamination of survival cultures is frequent,the collection of epithelia under proper conditions is difficult andfrequent errors take place when obtaining antigens due to epitheliacontamination or degradation. Therefore, costs per unit vaccine arehigh.

More recently, BHK (Baby Hamster Kidney, line 21, clon 13) cell culturewas introduced; cell cultures in suspension are used for virusreproduction.

Such a process requires the use of sophisticated devices in order tomaintain cell multiplication (Synthetic or semi-synthetic means with theaddition of fetal sera). Due to their high cost, fetal sera have beenreplaced by adult bovine serum, but the use of the latter causes therisk of neutralizing antibodies which, in many cases, completelyprevents virus reproduction. Further, cell adaptation to suspensionculture is required, as well as virus adaptation to its multiplicationin these cell cultures, with corresponding risks of antigenmodification. Lastly, expensive equipment is required in order toguarantee culture bacterial sterility.

The process of the instant invention, in the steps corresponding to theobtaining virus, is improved over the processes of the prior art,basically due to the fact that it transforms the whole animal into amedium appropriate for virus reproduction, this fact being valid alsofor adult rodents and ruminants.

In fact, prior to virus inoculation, the animal is completely irradiatedwith ionizing radiation in a dosage that is between a minimum value forassuring immunological depression and a maximum value for assuringanimal survival until its death caused by the action of the highlyproliferated virus. Optimum dosages were found to be between 700 and2700 rads (according to the species).

It was also found that a period of time of 24-36 hours betweenirradiation and virus inoculation was convenient in order to obtain thebest results.

Another important improvement is the use of antibiotics during the viralproliferation period, in order to avoid bacterial reproduction promotedby the immunodepression caused by radiations.

In this way, an economical, technologically simple, high quality viralantigen source is obtained, which also bears a low contamination riskand low antigen modification rates.

Since known methods of virus collection may be applied to some tissuesor cultures, the following collection step is preferred.

This step comprises removing skin and viscerae from the host animal.Soft parts are removed from the rest of the cadaver and are crushed withthe addition of saline solution; the mixture obtained should be visuallyhomogeneous.

Lastly, a liquid phase is separated by means of filtration orcentrifugation, thus obtaining the required active viral suspension.

In relation to virus inactivation, the known methods are based on thechemical action of some substances, such as acethylethylenimine,betapropiolactone, glycidaldehyde, ethylethylenimine, bis-ethylenimine(BEI) or formaldehyde. In such a process, the viral suspension should bekept at room temperature or higher temperatures for 24-48 hours.

Under these conditions, physical and chemical agents act to degrade thevirus antigenic proteins.

On the contrary, radiation with ionizing radiations, in the mentioneddosages, carried out at extremely low temperatures (typically -70° C.,and always below 0° C.) does not cause the mentioned enzymic reactions.

Virus radiation with ionizing rays is already known in the art, but notfor the purpose of the instant invention, but for forming part of thebasic investigations.

Ernest Pollard, U.S., has applied radiation for obtaining inactivationof some virus properties (Inactivation Theory).

Another known reference of prior art is that disclosed by WilliamGinoza, "Inactivation of viruses by ionizating radiations and by heat",Methods in Virology, IV, pages 139-205 (1968).

Nevertheless, none of the above references are related to the use ofradiations for obtaining industrial production of vaccine.

Ionizing radiation used for radiating animals as well as forinactivating the viruses may be of the electromagnetic radiation type,such as X-ray or Gamma-ray, or of the particle radiation type, such asalpha-particles, positions, electrons, protons, neutrons or deuterons.

Whichever its origin, foot and mouth disease antigen has a rather poorimmunological ability. Therefore the antigen should be improved by othersubstances capable of exerting immunity coadjutant action, in relationto power as well as to duration.

In accordance with known processes, aqueous or oily carriers may beused. The former include aluminum hydroxide and the duration of theimmunity obtained is not longer than four months. The second areemulsions, but in this case known vaccines have relatively largedroplets, and in some cases their stability is low, thus causing ruptureof the emulsion.

With the process of the present invention it is possible to obtain aminimum immunity term of six months, thus reducing the vaccineadministration to bovines to twice a year.

Therefore, the instant invention proposes a formulation comprisingproducing a "water in oil" type emulsion, the discontinous internalphase of which is constituted by the inactivated viral suspension andits external dispersion phase comprises a mineral oil.

The mineral oil, acting as immunity adjuvant, is of the liquid paraffintype, which is a mixture of different proportions of paraffins andnaphtenes, but lacking cyclic hydrocarbons due to their probablecarcenogenic action.

Another suitable oil is polybutene, which is an oily hydrocarbonprepared through polymerization of olefins, mainly comprised bymethylated isobutenes of constant formula.

Whichever the selected oil may be, when the antigen is to beconcentrated or the emulsion viscosity is to be lowered, the viralsuspension is previously adsorbed to the aluminum hydroxide with 2%aluminum oxide.

In both cases, and in order to obtain a low viscosity, stable,homogeneous emulsion of the type "water in oil", having small droplets(of a diameter not larger than 0.1 micron) and easy to be obtained at anindustrial scale, three emulgents are employed: a lipophilic emulgent,sorbitan sesquioleate of hydrophilic-lipophilic balance (HLB) 3.7; ahydrophilic emulgent, polyoxiethylene sorbitan mono-oleate, HLB 15, inthe case of an oil emulsion of the viral solution, or HLB 10 when theviral suspension is incorporated to aluminum hydroxide; and n-butanol asco-surfactant.

The object is to obtain any of the following formulations:

    ______________________________________                                        1.     Liquid paraffin  40.50%                                                       Sorbitan sesquioleate                                                                          6.50                                                         Polyoxiethylene sorbitan                                                                       1.00                                                         mono-oleate, HLB 15:                                                          n-butanol        2.00                                                         Inactive viral suspension                                                                      50.00                                                 2.     Polybutene       40.50                                                        Sorbitan sesquioleate                                                                          6.50                                                         Polyoxiethylene sorbitan                                                                       1.00                                                         mono-oleate, HLB 15                                                           n-butanol        2.00                                                         Inactive viral suspension                                                                      50.00                                                 3.     Liquid paraffin  53.60-43.60                                                  Sorbitan sesquioleate                                                                          2.00                                                         Polyoxiethylene sorbitan                                                                       2.40                                                         mono-oleate, HLB 10                                                           n-butanol        2.00                                                         Aluminum hydroxide plus                                                                        40.00-50.00                                                  inactive viral suspension                                              4.     Polybutene       53.60-43.60                                                  Sorbitan sesquioleate                                                                          2.00                                                         Polyoxiethylene sorbitan                                                                       2.40                                                         mono-oleate, HLB 10                                                           n-butanol        2.00                                                         Aluminum hydroxide plus                                                                        40.00-50.00                                                  inactive viral suspension                                              ______________________________________                                    

The steps involved in the process are the following:

(a) Mineral oil and emulgents are mixed into a tank at 4° C.;

(b) viral suspension, pure for formulations 1 or 2, or mixed withaluminum hydroxide in the case of formulations 3 or 4 (stirring for twohours, 1 part aluminum hydroxide and 1-5 parts of viral suspension, inaccordance with the concentration desired) is introduced into anothertank at 4° C., settling is allowed for 24 hours, and supernatant neededfor returning to the original volume of aluminum hydroxide is removed;

(c) contents of both tanks are poured into a closed and sterile loophomogenizator, operating under high pressure and continuous flow;

(d) the product is collected into a third tank.

SUMMARY OF THE INVENTION

In general, the above process for producing anti-foot and mouth diseasevaccine may be used for the production of other antiviral vaccines,provided some modifications are made, which do not affect the generalscope of the invention.

Therefore, an object of the instant invention is to provide a processfor preparing antiviral vaccines on an industrial scale with minimumrisks caused by contamination, bacterial proliferation or virusdegradation through physical or chemical action.

Another object of the invention is to provide a process for preparing alow cost antiviral vaccine.

Still another object of the invention is to provide a process forpreparing an antiviral vaccine suitable for different types of viruses,such as that producing foot and mouth disease.

A further object of the invention is to provide a process for preparingan antiviral vaccine, particularly an anti-foot and mouth diseasevaccine, wherein virus production is effected on adult animals, eventhose animals which are not naturally sensitive to the virus, such ashamsters, in almost all their organs and tissues.

Another object of the invention is to provide a process for preparing avaccine wherein, once the active viral suspension is obtained, the sameis inactivated by a method withstanding low temperatures and assuringits antigenic character.

Finally, another object of the invention is to provide a process forpreparing an anti-foot and mouth disease vaccine which provides a longerimmunity period.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting virus survival vs. radiation dose received;

FIG. 2 is a scheme of the arrangement used for radiating ovine andbovine cattle;

FIG. 3 shows some of the most important components of the viruses understudy; and

FIG. 4 is a survival semilogarithmic curve, in accordance with theTarget Theory, for different sizes of targets.

Table 1 lists viral proliferation results on previously radiatedanimals.

DETAILED DESCRIPTION OF THE INVENTION

Viruses irradiated with ionizing radiations may be fully destroyed byhigh doses, while, under controlled conditions, partial inactivation ofdifferent viral properties, such as hemoagglutination, antigenicity,uneffectiveness, etc. may be obtained.

The loss of infective ability of the virus thus obtained permits theproduction of suitable materials for vaccine preparation.

The conditions under which such material is obtained are establishedthrough the study of virus inactivation curves, which allowdetermination of the minimum dose to be applied for obtainingnon-infective materials, and which are obtained plotting the number ofunits surviving radiation vs. dosage. The curve thus obtained is knownas Survival Curve or Dose-Response curve and differ from other survivalcurves in that the latter may not be explained only in biologicalvariability terms.

The above gave rise to the interpretation of the results through theso-called "Impact Theory", also known a Target Theory, which widelyexplains different kinds of survival curves.

Considering the results obtained through investigation and experiments,inactivation curves not only depend on virus external factors, such asdosages, radiation rates, radiation type, temperature and nature of thesubstrate on which virus is hosted during the process, but also on thecharacteristics of the virus per se, such as its chemical composition,e.g. DNA or RNA nucleus, or on the nature of its molecular structuresuch as, for example, one or more single or double conjugated chains.

FIG. 1 shows the results obtained in the following cases:

(a) inactivation of Rauscher Murine Leukemia virus;

(b) inactivation of Herpes Simplex virus,

(c) inactivation of foot and mouth disease virus.

In all cases, the intersection of the curve with the X-axis gives thedose needed for inactivating 10⁶ times the original population, i.e.In/Fn=1.000.000 wherein In stands for Initial number and Fn for Finalnumber of viral particles after the process.

For foot and mouth disease virus, inactivation of the three typesthereof existing in Argentina (A. O. C) was carried out throughgamma-radiation, results similar to those shown on the graph beingobtained.

In all cases, foot and mouth disease virus inactivated by ionizingradiation fully retains its ability for fixing to the complement in thepresence of anti-foot and mouth disease hyperimmune serum.

With the viruses reproduced in irradiated animals, being the virusesinactivated by ionizing radiation, monovalent and trivalent experimentalanti-foot and mouth disease vaccines were prepared and tested for theirpower and compared to known vaccines. Similar or better results wereobtained.

Curve -a- in FIG. 1 represents the inactivation of Rauscher MurineLeukemia virus of which comprises two RNA complexed chains designed asR-MuLV and R-SFFV, radiated with gamma-rays.

Curves -b1- and -b2- represent inactivation of viral particles from theHerpes Simplex family designed HSV-1 (Thea) and HSV-2 (Muller), bothhaving DNA, radiated with gamma rays.

Curves -c1- and -c2- represent foot and mouth disease virus A (FMDV)inactivation, subjected to two types of radiation, namely gammaradiation from Co 60 and accelerated particles radiation (27 MeVdeuterons) from Syncrocyclotron at, Buenos Aires, respectively.

FIG. 2 shows the plan of the room in which ovine and bovine cattle wasirradiated with Co 60.

The equipment is a "Gamma beam 150" from the Radiobiology Center of theAgronomy and Veterinary Faculty, Buenos Aires University.

Reference numeral 1 designates cobalt source 60. The animal was locatedin a wooden container 2; free spaces were filled with a material similarto tissue to assure an even radiation.

The animal received a half-dose while its mean longitudinal lineremained following a 1.10 m radius circumference. The other half-dosewas received placing the animal in an inverted position, i.e. with theother side towards source 1.

Numeral 3 represents the access door and number 4 the command room.

Due to the importance of dosimetry for the correct result evaluation,checking of the dose was considered necessary. This was carried out witha thermo-luminiscent dosemeter introduced into the animal through oralor rectal route. This process gives a correct idea of the doseintroduced into the animal.

Table I shows the results obtained by virus inoculation in adult rodentsand previously radiated bovines.

When carrying out the process of the invention, those skilled in the artmay introduce modifications and/or improvements, all of which are to beconsidered within the scope of the instant invention which is onlydetermined by the spirit and scope of the appended claims.

                  TABLE I                                                         ______________________________________                                                        CONTROL METHODS                                                               Titration for organ                                                           mixture                                                       SPECIES USED      DOSE    minimum                                                                              maximum                                      AGE (weeks)       (rad)   (10 exponent)                                                                             C.F.                                    ______________________________________                                        Hamsters (Cricetus acureatus)                                                 8-11  R + V       800     7.1    9.4    ++++                                        R + V + A   800     7.9    10.4   ++++                                        R + V + A   800     10.4   12.5   ++++                                        (virus                                                                        passages)                                                               Pirbright Mice                                                                3-4   R + V       600     5.5    6.5    ++++                                        R + V       800     5.5    7      ++++                                  7-8   R + V       600     3.4    4.2    ++++                                        R + V       800     4.7    5.4                                          C3H/Ep. Mice                                                                  7-8   R + V       700     3.5    4.5    ++++                                                    900     5.4    6.4    ++++                                  Wistar rats                                                                   7-8   R + V       1,200   4.5    7.5    ++++                                  Ovines                                                                        24- 50                                                                              R + V + A   2,700   4.8    6.5    ++++                                  Affected muscle (mass proliferation of virus)                                 R + V + A     2,700   6.3      8.4    ++++                                    Ovines inoculated with virus serial groups                                    R + V + A     2,700   7.8      8.6    ++++                                    ______________________________________                                         Notes:                                                                        R: radiation                                                                  V: virus                                                                      A: antibiotic                                                                 F.C.: complement fixation                                                

The following is a plan for producing 1,000,000 doses monthly oftrivalent anti-foot and mouth disease vaccine (eventually tetravalentvaccine).

(1) Hamsters (Cricetus aureatus, 60-70 days old, male and female, in therequired amount (480 per month).

(2) The required amount (approximately 120 hamsters) are transferred tothe room in which radiation takes place and are subjected to a radiationdose comprised between 700 and 1200 rads.

(3) During the time comprised between 24 hours before radiation and thetime in which virus is inoculated, which is 24 to 36 hours after,radiation antibiotics are injected in to the animals (Gentamicine, 4mg/kg daily, in a single dose or divided into two doses).

(4) Animals are located in a laboratory room specially designed for thisend and into containers assuring asepsis, and fed with sterile food andwater.

(5) 24-36 hours after radiation, active virus is inoculated. Virus iscontained in bovine origin material, in the form of i.m. injectablesuspension.

(6) Animals are permanently checked in order to determine their death,upon which cadavers are collected and viscerae and skin are removedtherefrom, and are kept into freezers at -40° C.

(7) Material gathered (sufficient for 1,000,000 monovalent doses) withthe addition of saline solution, is crushed until a homogeneoussuspension is obtained, separated by filtration and centrifugation.(Prior to the last centrifugating operation, chloroform is added up to amaximum of 5% ).

(8) After a final filtration, the active viral solution is again frozenat -40° C. into plastic containers suitable for further inactivationthrough radiation.

(9) The above material is inactivated by radiation from cobalt beams insuitable containers and is kept frozen through contact with dry ice(-70° C.).

(9') Samples for titration and classification (types).

(10) Radiation dose for anti-foot and mouth disease vaccine is of 4Mrads.

(11) Vaccine formulation

Virus is diluted until titration is obtained, one titre of 10⁷ inlactant mice (from an original titration of 10¹⁰ to 10¹² for each viraltype).

Hydroxisaponinated vaccine: With the antigen prepared and inactivated bythe above technology, hydroxisaponinated vaccine lots are prepared inaccordance with classic methods, without virus concentration. Into asterile tank, at 4° C., 40% of aluminum hydroxide specially prepared tothis end with 2% of aluminum oxide is added, then 60% of the viralsuspension previously inactivated through radiation is added, containingequal parts of virus, i.e. each 5 ml dose has 1 ml of each antigen fortrivalent vaccine. Thimerosal 1:30000 is used as preserver. The mixtureis stirred for 2-4 hours, saponine is added (20 ml) of a 5% suspensionper liter of vaccine and 50 ml of glycerine per equal volume, asprotector.

(12) Oily vaccine:

The formulation of the oily vaccine comprises the following steps:

(a) placing the inactivated viral suspension into a first tank at 4° C.;

(b) mixing a mineral oil of the liquid paraffin type with sorbitansesquioleate HLB 3.7 as lipophilic emulgent and polyoxiethylene sorbitanmono-oleate HLB 15 as hydrophilic emulgent, into a second tank at 4° C.;

(c) pour the contents of the first and second tanks into ahomogenizator;

(d) homogenize, under high pressure, at 4° C., under continuous flow insterile closed loop, until a stable emulsion is obtained, of the type"water in oil" with homogeneous droplets of no more than 0.1 microndiameter.

(e) collect the emulsion into a third tank, this emulsion comprising theprepared vaccine. Preparation of oily vaccine/aluminum hydroxide.

(a') place the inactivated viral suspension into a first tank; mix saidsuspension with aluminum hydroxide, with stirring, for 2 hours, settlingduring 24 hours. Supernatant is removed in order to obtain the originalvolume of aluminum hydroxide.

(b') mix a mineral oil, liquid paraffin or polybutene, with emulgents:polyoxiethylene sorbitan sesquioleate HLB 10 into a second tank.

(c') proceed as in (c), (d) and (e).

The following steps were carried out for antigen control of virusesreproduced in radiated animals and inactivated through radiations:

(a) monovalent and trivalent experimental anti-foot and mouth diseasevaccines were prepared.

(b) these vaccines were tested for their power as compared with knowncommercial vaccines and the results obtained were the same or better.

Results obtained for anti-foot and mouth disease vaccine allowpredicting application of the process of the invention for preparingother antiviral vaccines, in light of the target theory, also calledimpact theory, of the practical results obtained.

Target theory may be applied to the inactivation of any type of simplebioacting molecule, such as an enzyme or complex micro-organisms such asviruses or bacteria, regardless their size.

Virus inactivation through radiations is based on the assumption of theexistence of a sensitive target which is destroyed when one or moreimpacts fall thereon.

In the case of viruses, this target is well determined and isconstituted by viral nucleic acid. Therefore, the viral target analysisalso constitutes the analysis of the nuclear structure, and this impliesnot only the chemical portion but also its molecular framework.

Impact interpretation is differentiated whether the radiation iselectromagnetic or corpuscular.

In the first case, the action is directed to a point, absorbed energyproducing rupture of a link is the ionizing energy, i.e. E=60 eV.

Int he case of corpuscular radiation, rupture is produced basicallythrough the action of a lower energy secondary particle sheath affectinga cross section of the particle path of the beam, which is carried outwith a lower energetic yield.

Target theory is based on two physical observations and a postulate:

(1) Ionizing radiations transmit their energy in discrete quantums.

(2) Interactions or impacts are independent from each other and followPoisson Law.

(3) The response under study takes place if the specific target hasreceived a particular number of impacts.

The target, with a volume v (cubic cm), may represent the sensitivestructural size of the radiated object. For mathematical purposes, v isonly a parameter measuring sensitivity to radiation.

Dose D may be expressed in terms of impacts per volume unit.

Product v×D represents the mean number of impacts within the volume vafter a dose D, the possibility existing of exactly occurring n impactsdue to Poisson distribution:

    P(n)=(v·D).sup.n ·e.sup.-v·D /n!

If n impacts are required for producing inactivation of an object, allobjects receiving n-1 impacts or less will survive.

Survival curve is obtained taking the summatory of all units whichreceived an impact number equal to 0, 1 2, . . . , n-1: ##EQU1## whereinN is the survival number and No the population prior radiation.

Theory extension to m targets of the same volume is expressed by themortality curve: ##EQU2## and the extension corresponding to m targentsof different volumes: ##EQU3##

From the above formulae, it is to be noted that, the greater the targetand with the same dose mortality is higher, which leads to theassumption that viruses greater than Picornavirus will have lowerinactivation doses.

In Table II other important formulae of the Target Theory are listed.

It is to be noted that doses in excess do no lead to the requiredresult, in what concerns to vaccine preparation. In fact, the object isto destroy nucleic acids but not to affect proteins which give raise tothe required antigenicity.

                  TABLE II                                                        ______________________________________                                        OUTSTANDING PARTICULAR RELATIONSHIPS                                          OF THE TARGET THEORY                                                          ______________________________________                                        1.     Survival curve for the case of: 1 impact - 1 target                           N/No = e.sup.-v.D                                                      2.     Survival curve for the case of: 1 impact - 1 target,                          two different sizes                                                           N/No = a · e.sup.-v1.D + (1 - a) · e.sup.-v2.D       3.     Survival Curve TITRATION-DOSE                                                 T = q · D                                                     4.     Survival line slope q.                                                 5.     Dose of 37% survival                                                          D.sub.37 = - log e/q                                                   6.     Target molecular weight                                                       MW.sub.t = Avogadro No./D.sub.37                                       7.     Target sensitive volume                                                       v = 1/D.sub.37                                                         ______________________________________                                    

Low temperatures are used during the virus inactivation step, since theyfreeze water in the saline solution, avoid the higher activity ofcertain chemical species in a liquid medium, even with not in excess.

As mentioned in connection with FIG. 1, three types of viruses have beentested, with different morphological features, thus embracing the wholespectrum of molecular weights and sizes, chemical composition andmolecular structure.

Foot and mouth disease virus was obtained from large stocks of stains"A", "C" and "O" existing in Argentina.

Rauscher virus was received from Dr. Komitowsky from the ExperimentalPathology Institute, German Cancer Investigation Center (DKFZ),Heidelberg, and was taken from original stocks from Bayer-Leverkussenlaboratories.

Herpes Simplex virus was received from Dr. Darai, Medical VirologyInstitute, Heidelberg University; it corresponds to strains HSV-1 (Thea)and HSV-2 (Muller).

The last two viruses pertain to oncogenic virus families, Class C, withRNA genome (Rauscher) and DNA genome (Herpes Simplex).

The outstanding morphological features of the viruses usd in the testsare summarized in the following Table III.

                                      TABLE III                                   __________________________________________________________________________                    FOOT AND MOUTH                                                CHARACTERISTICS DISEASE     RAUSCHER                                                                             HERPES                                     __________________________________________________________________________    Group           Picornavirus                                                                              Retrovirus                                                                           Herpesvirus                                Size (A)        250         800-1200                                                                             1800-2000                                  Virions morphology                                                                            bare        encapsulated                                                                         encapsulated                               a. coating                                                                    Virions morphology                                                                            icosahedron helical                                                                              icosahedron                                b. geometry                                                                   Capsomer No.     32         none   162                                               Molecular weight                                                                       2,7         10      68                                        Nucleic                                                                              × 10.sup.6 d                                                     Acid   Chain framework                                                                        simple RNA  simple RNA                                                                           double DNA                                        Ploidism haploid     diploid                                                                              conjugated                                 __________________________________________________________________________

FIG. 1 shows coincidence among the results obtained through theoreticalprovisions.

Survival curves based on experimentally determined doses correspond, inaccordance with the target theory, to the following two main types:

(a) In what concerns to foot and mouth disease virus and herpes virus,the relationship found is purely exponential, therefore, itssemilogarithmic representation vs. dose is a straight line. This isexplained by the existence of a single target, destroyed with a singleimpact (a single sensitive volume), corresponding to expression 1 ofTable II.

(b) Regarding Rauscher virus, straight line disruption is noted and theline adapts to the curve obtained in the model of 1 target and 1 impact,but of two different sizes. Expression 2 of Table II is applicable.

In all cases, value of 37% survival (D 37 dose) may be obtained throughexpressions 3, 4 and 5.

Upon knowing the above parameters, molecular weight MW and volume of thesensitive target may be calculated (Expressions 6 and 7).

Target size in expression 7 indicates that the mean impact value withinthe volume after dose D37 is 1.

The above is basically important since it means that the total impactnumber coincides with the number of radiated objects, thus allowingtransference of the particular dose in erg/gram to impact No./gram,taking into account the first postulate of the target theory.

In order to compute the target volume or its molecular weight, thefollowing equivalences were used:

(1) Average energy per impact E=60 (eV)

(2) Definition of ##EQU4## (3) Target volume

    v=0.96×10.sup.-12 /ρ·D37 (cm)

wherein ρ=density (g/cm³) and D 37 in rads

(4) Target molecular weight

    MW.sub.t =5.8×10.sup.11 /D37 (daltons)

wherein D 37 is in rads

FIG. 3 depicts some of the most important structural components ofviruses under study, related to the instant invention.

Relative sizes of the viruses may be seen in -A, -B- and -C-. In a scalein which the written width of a sheet corresponds to 1 micrometer:

-A-: foot and mouth disease virus

-B-: Rauscher Leukemia virus

-C-: Herpes virus

-A- represents a polyhedral structure and cubic symmetry virus. Amongothers, poliomyelitis virus is included in this group. -B- and -C-represent viruses bearing a more complex symmetry. Both areencapsulated, but interal components have different structures: one ishelical and the other is cubic, such as is the case with foot and mouthdisease virus.

In -D- the nuclear is represented in a larger scale, and corresponds tothat of 162 capsomers Herpes virus. Pentamers may be seen at the cornersand hexamer capsomers in the remaining sites.

-E- shows, in a larger scale, details of the formation of a pentamerfrom more elemental structural units, such as that shown in -F-.

These units, formed by one or more polypeptides, similar or not,constitute the antigen monomers the properties of which should bepreserved in the viral inactivation step. These chains are proteinswhich, in many cases, were able to isolate and correspond to units suchas VPI of foot and mouth disease, or such as P 18 and P24 of AIDS virus,and which have a molecular weight not higher than 24.000 daltons. On theother hand, this value corresponds to that generally assigned as maximumvalue to a structural protein with antigenic properties.

FIG. 4 is a graph listing the outstanding results permitting thecreation of a methodology useful for the preparation of viral vaccinesthrough nucleus radiation inactivation, preserving the antigenic abilitygiven by smaller size units.

Curve (A) represents survival of a protein with an average molecularweight of 12.000 daltons vs. gamma radiation dose. Curve (B) representssurvival of a protein with an average molecular weight of 24,000daltons, under the same conditions.

These curves were obtained using the expressions deducted through thetarget theory.

Although curve (B) corresponds to the dimmensions of the antigencommonly found in some viruses, such as that of foot and mouth disease,this means an extreme value of dimmensions representing a viral antigen,since these molecules are sometimes cleaved into two or more equalcomponents such as that shown with -F- in FIG. 3, or most important, thetarget volume sensitive to its antigenic properties represents aconsiderable smaller fraction than its total weight. Therefore, curve(A) is considered the most representative of structural viral antigengroup.

Percentage values of antigens affected by radiation doses equivalents tothose of viral inactivation (4 Mrads), vertical dotted line in figure 4,are 8% for the most probable curve (A), and 16% for curve (B) (damagelimit) of 24,000 daltons. Further, this curve corresponds to an extremedamage value produced by radiations, not only for viral antigens, butfor the whole particle of the same molecular weight treated byradiations under identical experimental conditions, such as thoseindicated when disclosing the target theory.

Curve (C) of 2.7×10⁶ daltons expresses inactivation of foot and mouthdisease virus through gamma radiation. As seen in FIG. 1, this is theradio-sensitive extreme value for viruses under study. This is in factdue to the corresponding with infective nucleus of single chain toribonucleic acid, under the most radioresistant conditions.

The same above reasons are the grounds for assuming that not only theseviruses under study, corresponding to a wide spectrum of morphologicaland structural features, will have a limit behaviour expressed by curve(C), but also most of the viruses will have the same behaviour whenexposed to electromagnetic radiation doses, i.e. will be at the left ofcurve (C). This is due to the fact that the existence of viruses withmolecular weights notably lower than those corresponding to picornavirushas not been demonstrated and, further, since viruses with highermolecular weights have been found just between curve (C) and the Y-axis.

Viral inactivation carried out with corpuscular radiations on the threeviruses under study allows determining their lower efficiency in thesame doses.

Foot and mouth disease virus was inactivated with 27 MeV deuterons, thusdemonstrating a biological efficiency of 0.7, which would indicate theneed of applying a higher dose than that used in the case ofelectromagnetic radiations, for the same inactivation value. This resultis respresented by curve -c2- of FIG. 1.

The other two virus irradiated with other particles: 10 MeV electrons,104 MeV alpha particles and 52 MeV deuterons gave a similar result, thusindicating a relative biological efficiency lower than 1, as in the caseof foot and mouth disease.

We claim:
 1. A process for preparing an antiviral viccine, paticularlyanti-foot and mouth disease vaccine, which process comprises the stepsof:(a) radiating an animal free of active virus with ionizingradiations; (b) innoculating active virus into said animal; (c)maintaining the animal under controlled sterile conditions and underantibiotic administration until a high viral proliferation is obtained;(d) after animal's death, cooling the cadaver; (e) withdrawing skin andviscerae from the animal cadaver; (f) crushing the soft portions of theremaining parts of the cadaver with the aid of a saline solution inorder to obtain a visually homogeneous mixture; (g) mechanicallyseparating an active viral suspension from said mixture; (h)transforming the active viral suspension into an inactive viralsuspension through the application of ionizing radiation on saidsuspensions, which are maintained at a temperature substantially lowerthan 0° C.; and (i) formulating the vaccine from said inactive viralsuspension.
 2. A process as claimed in claim 1, wherein a period ofsubstantially 24-36 hours elapses between animal radiation and activevirus animal inoculation.
 3. A process as claimed in claim 1,wherein:(a) the active virus used in animal inoculation is an injectablesuspension of active foot and mouth disease virus; (b) an anti-foot andmouth disease vaccine is formulated in the formluating step.
 4. Aprocess as claimed in claim 3, wherein the ionizing radiation dose ofthe animal radiating step is substantially comprises between 700 and2,700 rads.
 5. A process as claimed in claim 3, wherein the dose appliedwhen transforming the active viral suspension into an inactive viralsuspension is substantially of 2-6 Mrad, said suspensions beingmaintained at temperatures substantially lower than 0° C.
 6. A processas claimed in claim 4, wherein said animal is an adult rodent.
 7. Aprocess as claimed in claim 4, wherein said animal is a ruminant.
 8. Aprocess as claimed in claim 1, wherein the mechanical method employedwhen separating the active viral suspension from the visuallyhomogeneous mixture consists in the filtration of said mixture.
 9. Aprocess as claimed in claim 1, wherein the mechanical method used whenseparating the active viral suspension from the visually homogeneousmixture consists in the centrifugation of said mixture.
 10. A process asclaimed in claim 3, wherein the vaccine formulating step comprises:(a)placing the inactivated viral suspension into a first tank at 4° C.; (b)mixing a mineral oil with sorbitan sesquioleate HLB 3.7 as lipophilicemulgent; with polyoxiethylene sorbitan mono-oleate HLB 15 ashydrophilic emulgent, and with n-butanol as co-surfactant, in a secondtank at 4° C.; (c) pouring the contents of the first and second tanksinto a homogenizator; (d) homogenizing, under high pressure, at 4° C.,and under continuous flow, in sterile closed loop, until a stableemulsion, of the "water in oil" type, having homogeneous droplets of nomore than 0.1 micron in diameter is obtained; (e) collecting theemulsion into a third tank, thus obtaining the vaccine so prepared. 11.A process as claimed in claim 10, wherein:(a) when placing said inactiveviral suspension into a first tank, the suspension is mixed withaluminum hydroxide, stirred for 2 hours and settled for 24 hrs, then thesupernatant is removed until the original volume of aluminum hydroxideis attained; (b) when mixing a mineral oil with emulgents, said mineraloil is mixed with sorgitan sesquioleate HLB 3.7 as lipophilic emulgent,polyoxiethylene sorbitan mono-oleate HLB 10 as hydrophilic emulgent andn-butanol as co-surfactant.
 12. A process as claimed in claim 11,wherein said mineral oil is liquid paraffin without cyclic hydrocarbons.13. A process as claimed in claim 11, wherein said mineral oil ispolybutene.
 14. A process as claimed in claim 3, wherein the ionizingradiations used for radiating said animal are electromagneticradiations.
 15. A process as claimed in claim 3, wherein the ionizingradiations used for radiating said animal are particle radiations.
 16. Aprocess as claimed in claim 3, wherein the ionizing radiations used intransforming the active viral suspension into an inactive viralsuspension are electromagnetic radiations.
 17. A process as claimed inclaims 3, wherein the ionizing radiations used in transforming theactive viral suspension into an inactive viral suspension are particleradiations.